Pseudomonas aeruginosa (PA) is a frequent cause of infection in individuals with cystic fibrosis (CF) and in hospitalized patients. In addition to being common, this pathogen is associated with poor clinical outcomes, which are associated with persistence of PA bacteria despite appropriate administration of antibiotics with in vitro activity against the causative PA isolate. A better understanding of the mechanism of persistence is essential for development of novel approaches to treating PA infections and reducing their associated morbidity and mortality. Macrolide antibiotics, especially azithromycin (AZM), are highly concentrated within host phagocytes and other cell types, and are therefore highly efficacious against obligate intracellular bacteria such as Chlamydia, Rickettsia, and Legionella spp. However they lack appreciable in vitro activity against PA. Interestingly whereas most antibiotics perform less well in vivo against PA than in vitro, macrolides have the opposite characteristics: they are associated with improved clinical outcomes despite an absence of appreciable activity in vitro. Inhibition of bacterial virulence factors, prevention of biofilm formation, and anti-inflammatory activity have all been postulated to contribute to the efficacy of macrolides, but the actual mechanisms by which these agents improve outcomes remain unclear. Although classically viewed as an extracellular pathogen, PA can access the intracellular compartment of host cells. It has recently been shown that a small proportion of infecting PA bacteria are internalized by and persist within host cells, both phagocytes and non-professional phagocytes. The clinical implications of this internalization have not been explored. In this proposal, we will test the hypothesize that conventional antipseudomonal antibiotics, which penetrate poorly into mammalian cells, fail to kill the small proportion of PA bacteria that reside in the intracellular compartment during infection, thus allowing persistence of small numbers of bacteria despite seemingly appropriate antimicrobial therapy. We will further determine whether AZM, which is concentrated to high levels within mammalian cells, exerts antibacterial effects against internalized PA, allowing clearance of this intracellular reservoir and improved clinical outcomes. Completion of the studies outlined in this proposal will further our understanding of the mechanisms by which AZM improves outcomes in PA infection. This information will in turn inform optimal dosing strategies for AZM and lay the foundation for the development of novel macrolide antibiotics with enhanced activity against internalized PA. In addition to their translational value, these studies are also relevant to the study of microbial pathogenesis in that they will highlight the importance of a novel virulence mechanism of PA: enhancing disease by accessing the intracellular compartment of host cells. In this regard, this proposal will have implications for other extracellular pathogens that have a limited ability to invade host cells, such as Streptococcus pyogenes, S. pneumoniae, and Haemophilus influenzae. PUBLIC HEALTH RELEVANCE: Infections caused by the bacterium Pseudomonas aeruginosa fail to respond as predicted to conventional antibiotics. In contrast azithromycin, an antibiotic with no predicted activity against P. aeruginosa, has been associated with improved clinical outcomes. We propose that azithromycin eradicate the small proportion of P. aeruginosa bacteria that reside within host cells and which are protected from most conventional antipseudomonal antibiotics. We further propose that eradication of these intracellular bacteria leads to the improved outcomes associated with azithromycin therapy. Completion of this proposal will increase our understanding of how azithromycin improves outcomes in P. aeruginosa infections and suggest novel treatment strategies for P. aeruginosa as well as other bacteria.